The overall goal of this proposal is to develop image reconstruction schemes and instrumentation for tomographic imaging of green fluorescent proteins (GFP) in small animals. Small animal imaging has gained considerable importance in recent years as more and more animal models for human diseases have become available. Imaging of disease progression and effects of treatments in animals has many scientific and economical advantages, as sacrificing animals at various disease stages and performing necropsy and histopathological studies can be sharply reduced. Optical techniques have proven to be very valuable when applied to small animal imaging because of an abundance of optical markers that can target and visualize various disease related processes on the cellular and molecular level. However, to date most optical imaging studies have only explored whole animal surface imaging without tomographic reconstruction. Only a few groups including our team have presented first tomographic results that reveal three-dimensional fluorescent probe distribution in small animals. In these cases the image reconstructions were exclusively based on the diffusion model of light propagation in tissue, which is an approximation to the more generally applicable transport model. This has limited the application of such codes to studies involving fluorophores that emit in the near-infrared, where tissue absorption is smaller than in the visible spectrum. Furthermore, only steady-state instrumentation or frequency-domain devices with modulation frequencies smaller than 150 MHz were employed. While promising, these systems still suffer from cross-talk between absorption and scattering effects, limited spatial resolution, and limited accuracy in fluorescence absorption and lifetime determination. It is highly desirable to extend optical tomographic methods to shorter wavelengths, which would provide a means to image green fluorescence proteins that emit light in the visible spectrum. Using GFP and its derivatives, it is now possible to visualize nearly any protein of interest in any cell, tissue, or species. Researchers working at all levels of biology, such as single-molecule dynamics, protein trafficking within cells, organelle dynamics, and cell and tissue behaviors during development, cancer progression, and other diseases, are nowadays making use of GFP mostly in vitro studies. The availability of an optical tomography system for in vivo GFP imaging would have a significant impact on this large area of research. Themain hypothesis of this proposal is that limitations related to existing instrumentation for optical tomography can be overcome by an imaging system that uses a frequency-domain light propagation model that is based on the equation of radiative transfer (also called "transport equation"). Combined with instrumentation that allows for modulation frequencies higher than the currently available 150MHz, this will lead to better spatial resolution and reduced cross-talk between scattering and absorption effects. Furthermore, using a multi-wavelength and multi-frequency imaging system offers unique opportunities to reduce undesirable background signal due to autofluorescence. The overall goal of this proposal is to develop novel optical methods for volumetric imaging of small animals that are used in research to better understand and treat numerous diseases. For example, the imaging system could help visualize tumor location and growth. If successfully implemented the new imaging system will provide new insights into how various diseases, such as cancer, spread and affect the body.